Novel combination treatment for acute myeloid leukemia (aml)

ABSTRACT

The present invention relates to a pharmaceutical product comprising a) as a first component an inhibitor of the MDM2-p53 interaction; and b) as a second component cytarabine; as a combined preparation for the sequential or simultaneous use in the treatment of cancer, particularly AML.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.17/324,248, filed May 19, 2021, which in turn is a Continuation of U.S.patent application Ser. No. 16/794,991, filed Feb. 19, 2020, which inturn is a Continuation of U.S. application Ser. No. 15/101,258, filedJun. 2, 2016, which is a National Stage Application of PCT/EP2014/076063filed Dec. 1, 2014, which claims priority from U.S. Provisional PatentApplication No. 61/912,152, filed on Dec. 5, 2013. Each of the priormentioned applications is hereby incorporated by reference herein in itsentirety.

The present invention relates to a combination therapy for the treatmentof proliferative disorders such as cancer, in particular Acute MyeloidLeukemia (AML). More particularly, the present invention disclosescombinations of the current backbone therapy in AML, the compoundcytarabine (Ara-C), together with a compound which acts as an inhibitorof the MDM2-p53 interaction. It was surprisingly found that suchcombinations show a more than additive (synergistic) effect.

p53 is a tumor suppressor protein that plays a central role inprotection against development of cancer. It guards cellular integrityand prevents the propagation of permanently damaged clones of cells bythe induction of growth arrest or apoptosis. At the molecular level, p53is a transcription factor that can activate a panel of genes implicatedin the regulation of cell cycle and apoptosis. p53 is a potent cellcycle inhibitor which is tightly regulated by MDM2 at the cellularlevel. MDM2 and p53 form a feedback control loop. MDM2 can bind p53 andinhibit its ability to transactivate p53-regulated genes. In addition,MDM2 mediates the ubiquitin-dependent degradation of p53. p53 canactivate the expression of the MDM2 gene, thus raising the cellularlevel of MDM2 protein. This feedback control loop insures that both MDM2and p53 are kept at a low level in normal proliferating cells. MDM2 isalso a cofactor for E2F, which plays a central role in cell cycleregulation.

The ratio of MDM2 to p53 is dysregulated in many cancers. Frequentlyoccurring molecular defects in the p16INK4/p19ARF locus, for instance,have been shown to affect MDM2 protein degradation. Inhibition ofMDM2-p53 interaction in tumor cells with functional p53 should lead toaccumulation of p53, cell cycle arrest and/or apoptosis. MDM2antagonists, therefore, can offer a novel approach to cancer therapy assingle agents or in combination with a broad spectrum of other antitumortherapies. The feasibility of this strategy has been shown by the use ofdifferent macromolecular tools for inhibition of MDM2-p53 interaction(e.g. antibodies, antisense oligonucleotides, peptides). MDM2 also bindsE2F through a conserved binding region as p53 and activatesE2F-dependent transcription of cyclin A, suggesting that MDM2antagonists might have effects in p53 mutant cells with functional p53signaling.

Inhibitors of the MDM2-p53 interaction have been shown to induceapoptosis in the established human AML cell line MOLM-13, whichoverexpresses MDM2 (K. Kojima, et. al., Blood 2005, 106(9):3150-9). Ithas now been found that the combination of compounds of formula (I)together with Ara-C provide more than additive effects in disseminatedMOLM-13 AML model in immunocompromised mice. Compounds of formula (I)and their preparation are disclosed in WO2013/135648. These compoundsact as pro drugs of the compound

4-{[(2R,3S,4R,5S)-4-(4-Chloro-2-fluoro-phenyl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid (herein compound A).

Compound A is for example disclosed in U.S. Pat. No. 8,354,444 andWO2011/098398.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical product comprising a)as a first component an inhibitor of the MDM2-p53 interaction (also“MDM2 inhibitor”); and b) as a second component cytarabine; as acombined preparation for the sequential or simultaneous use in thetreatment of cancer.

The present invention further relates to a method of treating a patientsuffering from cancer, comprising administering to the patient thecombination as mentioned above.

The present invention also relates to a kit comprising a) a firstcomponent which comprises, as an active agent, an inhibitor of theMDM2-p53 interaction; and b) a second component which comprises, as anactive agent, the compound cytarabine.

In addition, the present invention relates to the use of an MDM2inhibitor and cytarabine for the treatment of cancer.

A yet further aspect of the present invention is the use of an MDM2inhibitor, and cytarabine for the preparation of a medicament for thetreatment of cancer.

In one embodiment, the inhibitor of the MDM2-p53 interaction is selectedfrom a compound of formula (I)

whereby the compounds of formula (I) are further specified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the antitumor efficacy of the compound I-B incombination with cytarabine on MOLM-13-luc.c4 (AML) tumor burden inSCID-beige mice, by quantification of bioluminescence.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of formula (I), and in particular I-A and I-B as disclosedherein, are polyethylene glycol (PEG) prodrugs of compound (A) that weresynthesized to provide the solubility required for an intravenous (iv)formulation of the active parent molecule compound (A). An ivformulation is desirable to ameliorate dose-limiting gastrointestinalintolerability and exposure variability, as well as to provide anacceptable route of administration for treatment of hematologicalmalignancies and for pediatric use.

The anti-tumor activity of once weekly iv administration of compound I-Band oral (po) compound (A) were compared as monotherapies and incombination with the AML standard of care, cytarabine (Ara-C), in theMOLM-13 model. Both compounds I-B and (A) elicited a significantincrease in lifespan (ILS) as monotherapies, with up to 37% ILS observedas compared to Vehicle control animals. Despite the lack of monotherapyactivity with Ara-C, it did significantly prolong survival incombination with compound I-B or (A), with maximum ILS of 54% or 68%observed, respectively. The synergistic effect demonstrated by thepresent data suggests that the combination of targeting MDM2-p53 with anMDM2 inhibitor and inducing S-phase arrest with cytarabine (Ara-C) maybe an effective therapeutic strategy for the treatment of AML. Thesedata also demonstrate that the efficacy of compound (A) can bemaintained by the prodrug approach using the compounds of formula (I),and in particular I-A and/or I-B.

Therefore, in one embodiment, the present invention relates to apharmaceutical product comprising a) as a first component an inhibitorof the MDM2-p53 interaction; and b) as a second component cytarabine; asa combined preparation for the sequential or simultaneous use in thetreatment of AML.

In another embodiment, the inhibitor of the MDM2-p53 interaction isselected from a compound of formula (I)

wherein n is from 3 to 70.

In one embodiment, n is from 30 to 60.

In another embodiment, n is from 40 to 50.

In yet another embodiment, n is 41, 42, 43, 44, 46, 47, 48 or 49.

In another embodiment the compound of formula (I) is:

4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, 2000). Thiscompound is designated herein as compound I-A.

In another embodiment the compound of formula (I) is:4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, 2200). Thiscompound is designated herein as compound I-B.

In yet another embodiment, the MDM2 inhibitor according to the presentinvention may be the compound (A). Within this embodiment, compound (A)is preferably provided as preparation for peroral administrationcomprising an amorphous solid dispersion, preferably a microprecipitated bulk powder (MBP), comprising compound (A) and a polymerwhich stabilizes compound (A) in its amorphous form, preferably HPMCAS.The peroral preparation is reconstituted immediately beforeadministration as a suspension in Klucel/Tween. Compound (A) can beprepared according to methods for example disclosed in U.S. Pat. No.8,354,444 or WO2011/098398. Dose selection in the current studies wasbased on previously determined optimal doses for compound (A) inpre-clinical (animal) and clinical (phase 1) trials.

The pharmaceutical products or methods according to the presentinvention are particularly useful in the treatment or control ofhematological tumors, such as leukemias, and especially for thetreatment of Acute Myeloid Leukemia (AML). They may also be useful inthe treatment of other cell proliferative disorders caused bydisregulation of the MDM2-p53 interaction, such as cancer, moreparticularly solid tumors such as, for example, breast, colon, lung,melanoma, prostate, kidney, head and neck, or sarcoma.

In one embodiment the present invention provides the presentpharmaceutical products and/or methods for the treatment of AcuteMyeloid Leukemia (AML).

In another embodiment the present invention provides the presentpharmaceutical products and/or methods for the treatment of cellproliferative disorders caused by disregulation of the MDM2-p53interaction, such as cancer, more particularly solid tumors such as, forexample, breast, colon, lung, melanoma, prostate, kidney, head and neck,or sarcoma.

Formulations of the compounds of formula (I) include those suitable fororal, nasal and/or parenteral or intravenous administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy.

In one embodiment, the compound of formula (I) is provided in a stablelyophilized formulation for intravenous administration comprising fromabout 0.1 mg to about 100 mg of compound (I), from about 10 mM to about100 mM of a buffering agent, from about 25 mg to about 125 mg of alyophilization bulking agent and an isotonicity builder. The resultantformulation should have a pH of about 5-7 via adjustment with HCl orNaOH.

In another embodiment, the compound of formula (I) is dissolved in 0.9%sodium chloride in sterile water by vortexing, then filtered thru afilter into a septum sealed vial for intravenous administration.

The term “buffering agent” as used herein denotes a pharmaceuticallyacceptable excipient, which stabilizes the pH of a pharmaceuticalpreparation. Suitable buffers are well known in the art and can be foundin the literature. Preferred pharmaceutically acceptable bufferscomprise but are not limited to histidine-buffers, citrate-buffers,succinate-buffers, acetate-buffers and phosphate-buffers, especially,Succinic acid (20-50 mM) and Phosphoric acid (10-50 mM). Most preferredbuffers comprise citrate, L-histidine or mixtures of L-histidine andL-histidine hydrochloride. Other preferred buffer is acetate buffer.Independently from the buffer used, the pH can be adjusted with an acidor a base known in the art, e.g. hydrochloric acid, acetic acid,phosphoric acid, sulfuric acid and citric acid, sodium hydroxide andpotassium hydroxide.

The preferred “bulking agent” is Trehalose dihydrate but lactose,sucrose, sorbitol, glucose, rafffinose, mannitol, dextran and lowermolecular weight amino acids such as glycine, valine and arginine etc.and other bulking agents described in the scientific literature may alsobe utilized.

As diluents for the formulated solution or reconstituted solution fromthe lyophilized powder the following diluents such as sodium chloride(0.9%), 5% Dextrose, water for injection, Lactated Ringers solution orhalf normal saline may also be used. It is to be appreciated that thebulking agent may also act as the isotonicity building agent.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, as well as the particular mode of administration.The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof a formula I compound which produces a therapeutic effect. Generally,out of one hundred percent, this amount will range from about 1 percentto about ninety-nine percent of active ingredient, preferably from about5 percent to about 70 percent, most preferably from about 10 percent toabout 30 percent. A typical formulation is prepared by mixing a compoundof the present invention and a carrier or excipient. Suitable carriersand excipients are well known to those skilled in the art and aredescribed in detail in, e.g., Ansel, Howard C., et al., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia:Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al.Remington: The Science and Practice of Pharmacy. Philadelphia:Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook ofPharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.

Cytarabine was purchased from Hospira, Inc. Lake Forest, IL 60045 USA,as sterile solution (100 mg/ml) for intravenous (iv) injection.

The compounds of formula (I) as well as cytarabine are administered intheir therapeutically effective amount. More particularly, the compoundsof formula (I), especially of formula I-A and I-B, are dosed in order todeliver a therapeutically active amount of compound A to a patient. Atherapeutically effective amount of a compound in accordance with thisinvention means an amount of compound that is effective to prevent,alleviate or ameliorate symptoms of disease or prolong the survival ofthe subject being treated. The determination of the amount of a prodrug, for example a compound of formula (I), such as I-A or I-B, inorder to deliver a desired amount of active principal, for example thecompound A, to a patient is within routine work of a person of ordinaryskill in pharmaceutical sciences. In one embodiment of the presentinvention the dose of prodrug of formula I-B of 437 mg/kg is equivalentto 100 mg/kg of parent MDM2 inhibitor of compound (A), due to 22.88%active compound loading in the prodrug.

The therapeutically effective amount or dosage of a compound accordingto this invention can vary within wide limits and may be determined in amanner known in the art. Such dosage will be adjusted to the individualrequirements in each particular case including the specific compound(s)being administered, the route of administration, the condition beingtreated, as well as the patient being treated. In general, in the caseof oral or parenteral administration to adult humans weighingapproximately 70 Kg, a daily dosage of about 10 mg to about 3,000 mg,preferably from about 80 mg to about 1600 mg of compound (A), should beappropriate, although the upper limit may be exceeded when indicated.The daily dosage can be administered as a single dose or in divideddoses, or for parenteral administration; it may be given as continuousinfusion.

In one embodiment, the present pharmaceutical products comprisecompounds of formula (I) characterized in that they are dosed in suchway as to deliver compound (A) in an amount of from about 50 to about3000 mg/day, or from about 80 to about 2500 mg/day, or from about 80 toabout 1600 mg/day, or from about 200 to about 1600 mg/day, or from about400 to about 1600 mg/day, or from about 400 to about 1200 mg/day, orfrom about 400 to about 1000 mg/day, or from about 400 to about 800mg/day, or from about 400 to about 600 mg/day for an administrationperiod of up to about 7 days, preferably up to about 5 days, on days1-7, or preferably days 1-5, of a 28 day treatment cycle, followed by arest period of from about 21 to about 23 days, preferably up to about 23days. The daily dosage, i.e. the amount of compound (A) expressed inmg/day, can be administered as a single dose (qd) or in two doses (BID).When two doses are given, they are preferably administered in equalamounts, once in the morning and once in the afternoon.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of about 120mg/day to about 1200 mg/day for an administration period of up to 5days, on days 1-5, of a 28 day treatment cycle, followed by a restperiod of 23 days. Doses are administered as a single dose (qd) or intwo doses (BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of from about400 to about 1200 mg/day for an administration period of up to 5 days,on days 1-5, of a 28 day treatment cycle, followed by a rest period of23 days. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of from about400 to about 800 mg/day for an administration period of up to 5 days, ondays 1-5, of a 28 day treatment cycle, followed by a rest period of 23days. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of from about400 to about 600 mg/day for an administration period of up to 5 days, ondays 1-5, of a 28 day treatment cycle, followed by a rest period of 23days. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of about 120mg/day to about 800 mg/day for an administration period of up to 5 days,on days 1-5, of a 28 day treatment cycle, followed by a rest period of23 days. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of about 120mg/day to about 600 mg/day for an administration period of up to 5 days,on days 1-5, of a 28 day treatment cycle, followed by a rest period of23 days. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of about 120mg/day to about 400 mg/day for an administration period of up to 5 days,on days 1-5, of a 28 day treatment cycle, followed by a rest period of23 days. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred.

In another embodiment, the present pharmaceutical products comprisecompounds of formula (I), I-A or I-B, characterized in that they aredosed in such way as to deliver compound A in an amount of about 120mg/day for an administration period of up to 5 days, on days 1-5, of a28 day treatment cycle, followed by a rest period of 23 days. Doses areadministered as a single dose (qd) or in two doses (BID). Within thisembodiment, compound I-B is preferred.

A therapeutically effective amount (or “effective amount”) of cytarabinein accordance with this invention means an amount effective to achievethe synergistic, i.e. more than additive effect as demonstrated by thedata disclosed herein (see e.g. FIG. 1 ). Since cytarabine is used asthe backbone therapy for AML for many years, a lot of information isavailable to the person of skill in the art, for example a clinicalphysician, about effective and tolerated doses in humans. It has forexample been found that cytarabine can be dosed as single agent in thetreatment of AML (induction regimen) in high amounts, such as amounts upto 3 g/m² (intravenous) over 2 hours every 12 hours days 1 to 6. Areview about the use of cytarabine in the treatment of leukemias is forexample provided in “Nicholas D. Reese, Gary J. Schiller; Curr HematolMalig Rep, 2013, 8:141-148.” In certain combination therapies (e.g.induction therapy of acute non-lymphocytic leukemia), the usualcytarabine dose in combination with other anti-cancer drugs is 100mg/m²/day by continuous iv infusion (Days 1-7) or 100 mg/m² iv every 12hours (Days 1-7). (see for example www.hospira.com).

Therefore, in one embodiment the present invention provides apharmaceutical product comprising, a) as an MDM2 inhibitor, a compoundof formula (I), I-A or I-B, wherein said compound is administeredintravenously (iv) once or two times per day on days 1 to 5, followed bya 23 days rest period, of a 28 days treatment cycle; and b) as a secondcomponent an effective amount of the compound cytarabine; as a combinedpreparation for the simultaneous or sequential treatment of cancer,preferably AML. Within this embodiment compounds I-A and I-B arepreferred and are dosed in such way as to deliver compound (A) in anamount of from about 50 to about 3000 mg/day, or from about 80 to about2500 mg/day, or from about 80 to about 1600 mg/day, or from about 200 toabout 1600 mg/day, or from about 400 to about 1600 mg/day, or from about400 to about 1200 mg/day, or from about 400 to about 1000 mg/day, orfrom about 400 to about 800 mg/day, or from about 400 to about 600mg/day. Doses are administered as a single dose (qd) or in two doses(BID). Within this embodiment, compound I-B is preferred. Also, withinthis embodiment, compounds of formula (I), I-A or I-B are dosed in suchway as to deliver compound A in an amount of about 120 mg/day to about1200 mg/day for an administration period of up to 5 days, on days 1-5,of a 28 day treatment cycle, followed by a rest period of 23 days; or

-   -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of from about 400 to about 1200        mg/day for an administration period of up to 5 days, on days        1-5, of a 28 day treatment cycle, followed by a rest period of        23 days; or    -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of from about 400 to about 800        mg/day for an administration period of up to 5 days, on days        1-5, of a 28 day treatment cycle, followed by a rest period of        23 days; or    -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of from about 400 to about 600        mg/day for an administration period of up to 5 days, on days        1-5, of a 28 day treatment cycle, followed by a rest period of        23 days; or    -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of about 120 mg/day to about 800        mg/day for an administration period of up to 5 days, on days        1-5, of a 28 day treatment cycle, followed by a rest period of        23 days; or    -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of about 120 mg/day to about 600        mg/day for an administration period of up to 5 days, on days        1-5, of a 28 day treatment cycle, followed by a rest period of        23 days; or    -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of about 120 mg/day to about 400        mg/day for an administration period of up to 5 days, on days        1-5, of a 28 day treatment cycle, followed by a rest period of        23 days; or    -   compounds of formula (I), I-A or I-B are dosed in such way as to        deliver compound A in an amount of about 120 mg/day for an        administration period of up to 5 days, on days 1-5, of a 28 day        treatment cycle, followed by a rest period of 23 days; or    -   compounds of formula (I), I-A or I-B are dosed two times a day        (BID) as 600 mg doses in order to deliver a total daily dose of        about 1200 mg of compound (A) to the patient.

Also, within these embodiments and if not explicitly otherwise stated,doses are administered as a single dose (qd) or in two doses (BID), andcompound I-A or I-B are the preferred compounds.

In another embodiment, cytarabine is combined with the compounds offormula (I), I-A or I-B according to the following regimens as requiredfor two specific patient populations, i.e.:

-   -   1) Patients with relapsed/refractory AML or with de novo AML who        have adverse features per European LeukemiaNet guidelines; or        patients who have had antecedent hematologic disorders which        have transformed to AML. Within this embodiment, cytarabine is        dosed at 1 g/m 2 as a single dose (qd) during 6 days as a 1-3        hour intravenous (i.v.) infusion.    -   2) Patients with adverse features per European LeukemiaNet        guidelines who are considered candidates for intensive        chemotherapy treatment with standard doses of cytarabine and        anthracycline (daunorubicin or idarubicin) regimen (“7+3        Induction regimen”). Within this embodiment, cytarabine is dosed        at 100-200 mg/m² daily during 7 days as a continuous intravenous        infusion+daunorubicin 45 to 60 mg/m²; or +idarubicin 12 mg/m²        intravenously daily for 3 days.

Within any of the embodiments 1) or 2) above, compounds of formula (I),I-A or I-B are dosed as defined herein before. Also, within any of theembodiments 1) or 2) above, compounds of formula (I), I-A or I-B aredosed in such way as to deliver compound A in an amount of from about120 to about 1200 mg/day; or from about 400 to about 1200 mg/day for anadministration period of up to 5 days, on days 1-5, of a 28 daytreatment cycle, followed by a rest period of 23 days. Within thisembodiment, compound I-B is preferred.

In yet another embodiment, the present invention provides a method forthe treatment of cancer, comprising administering to a patient in needof such treatment a pharmaceutical product as defined hereinbefore.Within this embodiment, the MDM2 inhibitor is preferably selected fromcompound I-A of I-B. Dosage forms, dosages and treatment schedules forcompounds I-A or I-B and cytarabine are preferably as described above.Also, within this embodiment the cancer is a solid- or non-solid tumor,preferably the cancer is Acute Myeloid Leukemia (AML).

In another embodiment, the present invention provides the use of acompound of formula (I), preferably I-A or I-B, and cytarabine for themanufacture of a medicament for the treatment of cancer, in particularAcute Myeloid Leukemia (AML).

In another embodiment, the present invention provides a pharmaceuticalproduct comprising, a) as a first component a compound of formula (I);and b) as a second component the compound cytarabine, both administerediv once or twice a day, as a combined preparation for the simultaneousor sequential use in the treatment of cancer; characterized in that thedose of the compound of formula (I) corresponds to a dose of compound(A) within the range from about 50 to about 3000 mg/day, or from about80 to about 2500 mg/day, or from about 80 to about 1600 mg/day, or fromabout 200 to about 1600 mg/day, or from about 400 to about 1600 mg/day,or from about 400 to about 1200 mg/day, or from about 400 to about 1000mg/day, or from about 400 to about 800 mg/day, or from about 400 toabout 600 mg/day.. Within this embodiment, the compound of formula (I)preferably is the compound I-A or I-B, the cancer is AML, the daily doseis from about 200 to about 1600 mg given once or twice a day, and thedosage regimen for the compounds I-A or I-B is on day 1 to 5, followedby a 23 days rest period of a 28 days treatment cycle. More preferably,within this embodiment, the compound of formula (I) is the compound I-B,the cancer is AML, the daily dose is about 1200 mg given once or twice(BID, 600 mg) a day on day 1 to 5, followed by a 23 days rest period ofa 28 days treatment cycle.

The invention is now further illustrated by the following accompanyingworking Example.

Example Materials and Methods

Animals

Female SCID beige mice (10/group), obtained from Charles RiverLaboratories (Wilmington, DE) were used when they were approximately8-12 weeks old and weighed approximately 20-25 grams. The health of themice was assessed daily by gross observation and analyses of bloodsamples taken from sentinel animals housed on shared shelf racks. Allanimals were allowed to acclimate and recover from any shipping-relatedstress for a minimum of 72 hours prior to experimental use. Autoclavedwater and irradiated food (5058-ms Pico Lab mouse chow, Purina Mills,Richmond, IN) were provided ad libitum, and the animals were maintainedon a 12 hour light and dark cycle. Cages, bedding and water bottles wereautoclaved before use and changed weekly. All animal experiments wereconducted in accordance with the Guide for the Care and Use ofLaboratory Animals, local regulations, and protocols approved by theRoche Animal Care and Use Committee in an AAALAC accredited facility.

Tumors

Parental MOLM-13 human AML cells were stably transfected with Luc2lentiviral particles for 24 hrs in the presence of Polybrene (8 ug/ml)and then selected in the presence of Blasticidin for 3 weeks.Subsequently, one clone was selected by single cell plating in thepresence of 0.1 mg/mL G418 and was designated MOLM-13.luc.c4. Thelentiviral Luc2 expression plasmid was constructed by incorporating Luc2gene (Promega) in to pLOC lentiviral plasmid backbone (Thermo FisherScientific). Luc2 lentiviral particles were prepared by usingTrans-Lentiviral Packaging System (Thermo Fisher Scientific) asrecommended.

MOLM-13.luc.c4 was maintained with RPMI 1640 with L-glutamine (2 mM)media (GIBCO/Invitrogen, Carlsbad, CA) supplemented with 10%heat-inactivated Fetal Bovine Serum (HI-FBS; GIBCO/Invitrogen, Carlsbad,CA), and 1% 100 mM sodium pyruvate. Freshly dissociated MOLM13-Luc.c4cells (1×106 or 5×106) suspended in Phosphate Buffered Saline (PBS) werethen intravenously inoculated via the caudal tail vein into femaleSCID-beige mice.

Test Agent

4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, 2200), i.e.compound I-B, was dissolved in sodium chloride in sterile water byvortexing. It was then filtered thru a 0.22 micron filter into a septumsealed vial for intravenous administration. Dose of the drug of 437mg/kg is equivalent to 100 mg/kg of parent MDM2 inhibitor due to 22.88%active compound loading in the prodrug. Stock cytarabine (Ara-Cinjection, 100 mg/ml) was diluted in sterile 0.9% sodium chloride to 44mg/ml according to manufacturer's instructions and dosed at 200 mg/kg ivtwice weekly.

Compound I-B and cytarabine were administered iv using a lcc syringe and26-gauge needle at 437 mg/kg (9 ml/kg) weekly (q7d) and 200 mg/kg (4.5ml/kg) bi-weekly (2×/week), respectively. On days of concomitantadministration, compound I-B was dosed in the morning and cytarabine wasadministered 6 hours later in compliance with IACUC regulations forintravenous volume administration. Treatment duration was 3 weeks.

Monitoring

For increased life span (ILS) assessment, animal body weights weremeasured two to three times per week, and animals were monitored dailyfor any clinical signs of toxicity or excessive tumor burden (i.e. hindlimb paralysis or morbidity). In addition, progression of disease wasmonitored by in vivo bioluminescent imaging (BLI) using IVIS® Spectrumsystem. For each BLI session, mice received 100 mg/kg D-luciferin(Caliper Life Sciences/Perkin-Elmer) via ip injections and were imagedat 20 min post luciferin injection at either a 5 s or a 10 s exposuretime. Images were captured by the IVIS® Spectrum system and data werecollected and analyzed with Living Image 4.2.0 software (Caliper LifeSciences, Hopkinton, MA). Total photon fluxes (ph/s) representingluciferase activity within each fixed region of interest (ROI) coveringwhole tumors of individual mice were determined. The actual images ofmice are not disclosed herein. Data for quantification ofbioluminescence originated from this monitoring are provided in FIG. 1 .

Calculations & Statistical Analysis

Weight loss was graphically represented as percent change in mean groupbody weight, using the formula: ((W−W₀)/W₀)×100, where ‘W’ representsmean body weight of the treated group at a particular day, and ‘W₀’represents mean body weight of the same treated group at initiation oftreatment. Maximum weight loss was also represented using the aboveformula, and indicated the maximum percent body weight loss that wasobserved at any time during the entire experiment for a particulargroup. Toxicity is defined as ≥20% of mice in a given groupdemonstrating ≥20% body weight loss and/or death.

Quantification of bioluminescence allowed for direct longitudinalcomparison of tumor burden between treatment groups prior to surrogatedeath end points being reached. Tumor burden was graphically representedas the mean BLI photon flux+standard error of the mean (SEM), and mediansurvival was determined utilizing Kaplan Meier survival analysis.Statistical analysis of comparisons between groups was analyzed bytwo-way ANOVA, and post-hoc Bonferroni test (GraphPad Prism, version4.3). Differences between groups were considered to be significant whenthe probability value (p) was ≤0.05.

For survival assessment, morbidity or hind limb paralyses were monitoredas end points and results were plotted as the percentage survivalagainst days after tumor implant (GraphPad Prism, version 4.3). Hindlimb paralysis, morbidity or ≥20% body weight loss were used assurrogates for death. The % ILS was calculated as 100×[(median survivalday of treated group−median survival day of control group)/mediansurvival day of control group]. Median survival was determined utilizingKaplan Meier survival analysis. Survival in treated groups was comparedwith the vehicle group by Log-rank (Mantel-Cox) Test (GraphPad Prism,version 4.3). Differences between groups were considered significantwhen the probability value (p) was ≤0.05.

Results

Toxicity

Toxicity as assessed by animal body weight loss or gross clinical signswas not observed in the current studies. There were however, sporadicdeaths directly after iv dosing of compound I-B of undetermined cause(potentially technical, though unproven, see Table 1).

TABLE 1 Summary of Toxicity data % Change # of in Body animals Reasonfor Weight at ≥20% body Mortality/ Group Frequency Route Day 17 weightloss Mortality Morbidity Vehicle Control q7d + iv + iv −4.9 0 0 N/A2×/wk compound I-B q7d iv −2.8 0 1 Un- 437 mg/kg determined Ara-C 2002×/week iv −5.8 0 0 N/A mg/kg compound I-B q7d + iv + iv −6.1 0 2 Un-437 mg/kg + 2×/week determined Ara-C 200 mg/kg

Antitumor Efficacy and Assessment of Survival/Increase in Life Span(ILS)

Mice were inoculated with 5 million cells and drug treatment wasinitiated on day 3. BLI demonstrated significantly reduced photon countsfor mice receiving compound I-B monotherapy, whereas cytarabine (Ara-C)by itself showed no difference compared to Vehicle-treated control mice(see FIG. 1 ). These apparent reductions in tumor burden as assessed byBLI did translate into significant increases in lifespan, with 37% ILSobserved for groups treated with q7d 437 mg/kg compound I-B. Cytarabine(Ara-C) demonstrated a lack of antitumor activity as assessed by tumorburden (BLI) or ILS. On the other hand, combinations with Ara-C andcompound I-B elicited statistically significant % ILS as compared withVehicle control or monotherapy arm, demonstrating a clear enhancement ofantitumor activity in combination. These data are summarized in Table 2below, also including a comparison with orally administered compound(A).

TABLE 2 Summary of Efficacy data Cpd (A) Ara-C Cpd I-B Cpd (A) Cpd (A)Cpd I-B Cpd (A) 80 200 437 100 80 437 100 mg/kg mg/kg mg/kg + mg/kg +mg/kg + Vehicle mg/kg mg/kg qd × 5 2×/wk Ara-C 200 Ara-C Ara-C GroupControl q7d iv q7d po po iv mg/kg 200 mg/kg 200 mg/kg Median 20.5  28 25  28 20  31.5  26  34.5 survival (days) % ILS — *37 *22 *37  0 *†54  *†27 *†68   vs. Vehicle control *p < 0.05 Vs. Vehicle Control †p < 0.05Vs. Monotherapy Arms Cpd = compound

1. A pharmaceutical product comprising a) as a first component aninhibitor of the MDM2-p53 interaction; and b) as a second componentcytarabine; as a combined preparation for the sequential or simultaneoususe in the treatment of cancer.
 2. The pharmaceutical product accordingto claim 1, wherein the inhibitor of the MDM2-p53 interaction isselected from a compound of formula (I)

wherein n is from 3 to
 70. 3. The pharmaceutical product according toclaim 2, wherein n is from 30 to
 60. 4. The pharmaceutical productaccording to claim 2, wherein n is from 40 to
 50. 5. The pharmaceuticalproduct according to claim 2, wherein n is 41, 42, 43, 44, 46, 47, 48 or49.
 6. The pharmaceutical product according to claim 1, wherein theinhibitor of the MDM2-p53 interaction is4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, ˜2000).
 7. Thepharmaceutical product according to claim 1, wherein the inhibitor ofthe MDM2-p53 interaction is4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, ˜2200).
 8. Thepharmaceutical product according to claim 1 for the treatment of AcuteMyeloid Leukemia (AML).
 9. The pharmaceutical product according to claim1 for the treatment of solid tumors.
 10. A method for the treatment ofcancer, comprising administering to a patient in need of such treatmentan effective amount of a pharmaceutical product according to claim 1.11. The method according to claim 10, wherein the cancer is AcuteMyeloid Leukemia (AML).
 12. The method according to claim 10, whereinthe cancer is a solid tumor.
 13. (canceled)
 14. A pharmaceutical productcomprising, a) as a first component a compound of formula (I) accordingto claim 2; and b) as a second component the compound cytarabine,wherein both components are administered intravenously, as a combinedpreparation for the simultaneous or sequential use in the treatment ofcancer; characterized in that the dose of the compound of formula (I)corresponds to a dose of compound (A)

within the range from about 200 to about 1600 mg/day.
 15. Thepharmaceutical product according to claim 14, wherein the compound offormula (I) is the compound4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, ˜2000).
 16. Thepharmaceutical product according to claim 14, wherein the compound offormula (I) is the compound4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoicacid 1-mPEG-carbonyloxy-ethyl ester (mPEG, average MW, ˜2200).
 17. Thepharmaceutical product according to claim 14, wherein the compound offormula (I) is administered on days 1 to 5, followed by a 23 days restperiod of a 28 days treatment cycle.
 18. The pharmaceutical productaccording to claim 17, wherein the compound of formula (I) isadministered once daily (qd) or two times a day (BID).
 19. Thepharmaceutical product according to claim 14, wherein the cancer isAcute Myeloid Leukemia (AML).
 20. (canceled)